This
is one of many exercises available fromInvertebrate
Anatomy OnLine,an
Internet laboratory manual for courses in Invertebrate Zoology.Additional
exercises, a glossary, and chapters on supplies and laboratory techniques are
also available at this site.Terminology
and phylogeny used in these exercises correspond to usage in the Invertebrate
Zoology textbook by Ruppert, Fox, and Barnes (2004).Hyphenated
figure callouts refer to figures in the textbook.Callouts
that are not hyphenated refer to figures embedded in the exercise. The glossary
includes terms from this textbook as well as the laboratory exercises.

Chordata
is characterized by a suite of apomorphies including a dorsal hollow nerve cord,
notochord, pharyngeal gill slits, and a post anal tail (Fig 29-1).The
ancestor was a fishlike deuterostome that swam using alternating contractions of
right and left longitudinal axial muscles to create undulations of the body.The
flexible, incompressible notochord prevented these contractions from compressing
the body while allowing lateral deflection.The
chordate central nervous system is a hollow, median, longitudinal nerve cord
formed in the embryo by an invagination of surface ectoderm whose original
function was probably sensory reception. Paired pharyngeal gill slits connect
the lumen of the pharynx with the exterior and originally functioned in
suspension feeding with respiration being added later.A
muscular tail posterior to the anus is, although commonplace in chordates, an
unusual feature not found in other taxa.It
is an extension of the axial musculature and is the chief locomotory organ. An
additional apomorphy is the endostyle, a region of pharyngeal endoderm, that
secretes iodated compounds, either mucus or hormones.

Cephalochordata

Cephalochordates
retain many of the features of the ancestral chordate including the dorsal
hollow nerve cord, notochord, postanal tail, and pharyngeal gill slits used for
filter feeding.The
swimming and feeding modes are like those hypothesized for the ancestor.

Introduction

As
the most vertebrate-like of invertebrates, cephalochordates are studied in
either vertebrate or invertebrate zoology courses.Comparative
vertebrate anatomy traditionally begins with a consideration of cephalochordates
and other protochordates and invertebrate zoology often ends with them.Protochordates
are the inver­tebrate members of Chordata and include urochordates (sea
squirts), and cephalochordates (amphioxus, lancelets). These taxa are clearly
related to vertebra­tes but also show unmistakable relationships with other
invertebrates, most notably echinoderms and hemichordates.These
then are the taxa that establish the evolutionary connections between the
vertebrates and rest of the deuterostomes and ultimately, the remainder of the
animal kingdom.Cephalochordates
probably diverged from the evolutionary line leading to the vertebrates before
the end of the Precambrian.

Of
the protochordate taxa, it is Cephalochordata that is the most vertebrate-like
(Fig 29-32).These
fishlike marine animals have the expected chordate characteristics of dorsal
hollow nerve cord, pharyngeal gill slits, notochord, and post-anal tail.Although
they lack the brain, primary sense organs and sense capsules, braincase, bones,
kidneys, heart, an endothelial lining of the blood vessels, and vertebrae, which
characterize most vertebrates, they share many other features with vertebrates.Cephalochordates
are laterally compressed and fishlike in general morph­ology, have segmentally
arranged axial (body) muscles, and a vertebrate-like hemal system.As
you study these animals, watch for other features that remind you of
vertebrates.

Cephalochordata
is a small taxon of 30 species in only two genera,BranchiostomaandEpigonichthyes(=Asymmetron).Cephalochordates
are generally known by the common name "lancelet" and those used in laboratories
are usually referred to as "amphioxus", from a now obsolete generic name.Amphioxus
means “sharp at both ends, an apt name for these animals as you will soon see.
All species are less than 10 cm in length and most are closer to five or less.They
are segmented, coelomate deuterostomes.

Lancelets
inhabit shallow offshore sands in temperate oceans with only the head protruding
above the sand surface (Fig 29-4).They
are capable of swimming briefly using lateral undulations of the body caused by
contractions of the axial musculature.They
are efficient burrowers, entering the sand head first, employing side-to side
undulations of the body similar to those used in swimming.Lancelets
are harvested for human consumption in some parts of the Orient.

Cephalochordates
are filter feeders that use cilia to generate a current of water and
phytoplankton particles into the mouth (Fig 29-6).Water
and food enter the pharynx where they are separated by filtra­tion.The
water, but not the food, passes out through the gill slits and thus leaves the
gut cavity.It
then enters a surrounding water space, the atrium, and moves from there to the
exterior.Most
vertebrates have no structure like the atrium so it may be difficult to
visualize this relationship at first.Urochordates,
on the other hand, have exactly the same filter feeding apparatus (Fig 29-23).
The food particles are too large to pass through the gill slits and must remain
in the lumen of the pharynx from which cilia move them posteri­orly into
digestive and absorptive regions of the gut.The
original function of the perforated pharynx (gill slits) of the chordates is
thought to have been filter feeding with gas exchange being added much later in
chordate evolution.More
complete discussions of the biology of cephalochordates may be found in Ruppert
(1997), Ruppert, Fox, and Barnes (2004), Parker and Haswell (1921), Nash (2002),
and Jollie (1973).

Anatomy

1.Amphioxus:Adult
specimen(Fig 1, 2).Begin
your study with a preserved adult animal in a small dish of tapwater.Handle
the specimen carefully as the preservative renders them brittle.Place
the dish on the stage of your dissecting microscope and use a teasing needle to
manipulate it.You
will study only the external anatomy of this specimen and will not dissect it.Later
you will study the internal anatomy using wholemounts and cross sections.

Theintegument(=
skin) is the outer covering (Fig 29-3D).It
includes a monolayered, nonciliated epidermis with a basal lamina.There
is no secreted extracellular cuticle. Below the epidermis is a thick,
collagenous, translucent, gelatinous, connec­tive tissue dermis.In
life the entire animal is translucent but preserved specimens are opaque.

Note
the fishlike (fusiform or lanceolate) shape of the animal (Fig. 1, 29-3A).The
body is divided into head, trunk, and tail. Thehead,
at the anterior end, is small and poorly defined.Therostrumextends
anteriorly and overhangs the mouth and buccal cavity (Fig 2, 29-5).The
largemouthlies
under the rostrum and opens into a spaciousbuccal
cavity.The
mouth is surrounded by a ring of tentacle-likebuccal
cirri(=oral cirri).These
are involved in preliminary mechanical sorting of food particles and are
probably chemoreceptive as well. The roof and walls of the buccal cavity form
theoral hood.

Most
of the body is thetrunk(Fig
1, 29-3B), which extends posteriorly from the head to the anus (the anus will be
easier to see later when you study a cleared wholemount).The
trunk contains most of the gut, including the large conspicuous pharynx (not
visible at present), and the musculature. The segmental arrangement of theaxial
musculature(body
musculature) is readily apparent through the translucent integument.The
muscles are arranged in 50-75 V-shaped segmental bundles calledmyomeres.Successive
myomeres are separated from each other by connective tissue partitions calledmyosepta.The
segmental organization of animals is referred to asmetamerismand
the vertebrates, as well as several other phyla of animals, such as annelids and
arthropods, are said to bemetameric.Myomeres
on opposite sides of the body are asymmetric and out of register with each other
(Fig 3).

There
are no paired appendages but on either side of the trunk is a ventro-lateral
longitudinal ridge, themetapleural
fold(Fig. 1, 3).These
ridges run from the oral hood to a position just posterior to the gonadal
region.The
atrium, which is not visible in whole specimens, opens to the exterior via theatriopore,
located on the midventral margin at the point where the two metapleural folds
join the ventral margin.Farther
posteriorly, beside a slight dip between the ventral fin and the caudal fin, is
theanus, located
slightly to the left side of the ventral midline.The
anus is the posterior external opening of the gut and marks the posterior limit
of thetrunk.The
region of the body posterior to the anus is thetail.One
of the characteristics of chordates is the presence of this postanal tail.

A
posteriorcaudal fin(=tail
fin) extends around the dorsal and ventral margins of thetail.There
is a longdorsal finalong
most of the dorsal margin of the body.A
shortventral finis
located on the ventral margin of the trunk just anterior to the caudal fin.It
extends from atriopore to anus.

Figure 1.
JuvenileBranchiostoma,
cleared specimen. Cephchord16L.gif

Segmentally
arranged, paired, rectangular swellings along the ventral margins of the
myomeres are about 26 pairs of segmentalgonads(Fig
29-3A,B).Branchiostomais
gonochoric.The gametes
are shed into the atrium and are carried to the exterior by the water passing
through it and out the atriopore.

2.
Amphioxus: wholemount of juvenile specimen (Fig 1, 2).Continue
your study of with a wholemount of a cleared juvenile amphioxus.These
specimens are much smaller than the mature specimen and are sexually immature.Mature
individuals are too large and thick for examination with a compound micro­scope.Juveniles
are identical to adults except for their size and lack of gonads.Use
of a small cleared specimen permits examination of many features of the internal
anatomy without dissection.More
detailed structures and relationships will be studied at higher power using
cross sections.

Place
the slide on the stage of the compound microscope so that the animal is upside
down when viewed with the unaided eye.It
will, of course, be right side up when observed through the microscope.Study
the animal using 40X, and occasionally 100X, as appropriate.You
will see many features familiar to you from your study of the adult specimen.Relocate
and re-identify therostrum,head,
dorsal fin, caudal fin, ventral fin, tail, oral hood, buccal cirri,metapleural
folds, andatriopore.

Look
at the dorsal region of the animal and find thedorsal
fin, recognizable by its faint verticalfin
rays.The
fin is composed of a longitudinal series of coelomic spaces each known as afin
box(Fig 2, 29-5B).The
fin rays are the septa between successive fin boxes.Follow
the dorsal fin anteriorly and posteriorly noting that it extends along most of
the dorsal midline.

Figure 2.
Anterior end of a cleared juvenileBranchiostoma.
Cephchord17L.gif

Using
100X and careful focusing, you should be able to see themyomeresthat
make up most of the body.Themyoseptaappear
as faint, oblique, V-shaped lines.The
myomeres and myosepta extend ventrally to the metapleural folds but they are
hard to see against the confusing background in this region.Myomeres
are derived from segmental coelomic compartments and the muscles are derived
from the epitheliomuscular cells of the coelomic mesothelium (peritoneum).

The
dorsal hollownerve cordextends
most of the length of the body (Fig 1, 2).It
is usually pinkish in these preparations and has a distinctive, irregu­lar,
longitudinal row of black, light-sensitive, pigment cupocellirunning
along its ventral margin.These
ocelli face in various directions, some dorsal and some ventral.The
nerve cord extends anteriorly to about the base of the rostrum where it bears a
larger, terminal ocellus known as theeyespot.The
nerve cord extends posteriorly into the base of the tail.

Although
you will not see them, the nerve cord gives rise to paired segmental dorsal and
ventral nerves, homologous to the spinal nerves of the vertebrates.Paired
sensory and visceral motor nerves connect the nerve cord but somatic motor
neurons are absent, or nearly so.Instead,
the axial muscles and the cells of the notochord are innervated by cytoplasmic
processes of the muscle cells extending to the nerve cord.The
nerve cord is surrounded by a connective tissue sheath.

The
cavity of the hollow nerve cord is theneurocoel.
Anteriorly, the neurocoel opens to the exterior by a permanent, dorsal neuropore
at the base of the rostrum (Fig 29-5B). Chemoreceptors in the neuropore monitor
the water in its vicinity.The
lumen of the nerve cord (neurocoel) is expanded anteriorly to form a vesicle
sometimes referred to as the brain.

Ventral
to the nerve cord is thenotochord.It
is longer, relative to the length of the body, in these animals than in any
other chordate.It
is longer than the nerve cord and extends well into the rostrum, presumably as
an adaptation to facilitate digging into sand.The
appellation "Cephalochordata" for these animals alludes to the presence of the
noto­chord in the head.In
vertebrates the notochord extends anteriorly only as far as the middle of the
brain (mesencephalon).The
notochord is usually yellowish in these preparat­ions and appears to be
vertically striated.It
is composed of large, vacuolated, disklike epitheliomuscular cells arranged in a
stiff longitudinal column and surrounded by a thick connective tissue sheath
(Fig 29-4A,E).

The
notochord resists the deformation of the body that would otherwise result from
contraction of axial muscles. Embryologically, the notochord arises from a
dorsal median thickening of the roof of the archent­eron. The coelom of
enterocoelic animals arises from paired dorso-lateral out­pocketings of the
archenteron roof.

The
remainder of the animal is mostly digestive system.Find
once more thebuccal cirrisurrounding
theoral hood(Fig
29-5).Thebuccal
cavityis the gut lumen
within the oral hood.The
posterior wall of the buccal cavity is the transverse, muscularvelum.An
aperture, of adjustable diameter, in the center of the velum connects the buccal
cavity with the pharynx.Anterior
to the velum, the walls of the buccal cavity bear a series of thick ciliated
grooves which make up thewheel
organ.The
cilia in these grooves trap food particles in mucus for digestion farther
posterior in the gut. On the posterior side of the velum, the mouth is
surrounded by slender sensoryvelar
tentacles.

On
the dorsal midline of the buccal cavity is a deep ciliated fossa called
Hatschek's pit or Hatschek’s nephridium (Fig 29-5B).This
is an unpaired kidney whose duct opens into the anterior pharynx. It is
difficult to avoid attempting to homologize this structure with Rathke`s pouch
of vertebrate embryos and thus with the anterior pituitary of adult
verte­brates.Like
those structures, it is in the ectoderm of what amounts to the stomodeal region
and it extends dorsally toward the anterior end of the nerve cord.Definite
homology with the pituitary has not been demonstrated but structural and
positional criteria support the supposition.Hatschek's
pit is also secretory and releases mucus to entrap food particles.

Thepharynx,
as in most protochordates, is by far the largest, most conspicuous, and most
distinctive region of the gut (Fig 29-6, 29-3C).It
begins just posterior to the velum and is a large, intensely red-staining, oval
structure with numerous, narrow, obliquegill
slitsseparated by narrow
tissuegill bars(Fig
29-5B).The
gill bars are supported by a collagenous branchial skeleton presumably
homologous to the visceral (gill) skeleton of vertebrates.The
gills function primarily in filter feeding with the responsibil­ity for gas
exchange being met by the thin epidermis of the general body surface.It
is worthy of note that in larvae the number of gill slits equals the number of
myomeres.This
is consistent with the hypothesis that that the gill slits, like the myomeres,
were segmental structures in the ancestors of vertebrates.

The
pharynx is surrounded by a large water chamber, theatrium,
which is an invagination of the surface ectoderm (Fig 1, 3, 29-3C).The
atrium occupies most of the space between the pharynx and the body wall.It
is U-shaped in cross section and encloses the pharynx on all sides except
dorsally.This
spatial relationship will become clearer when you study the cross section slide.
Although the atrium may seem to be an unusual adaptation entirely absent in the
vertebrates, it is actually identical to the opercular cavity of larval anurans
(tadpoles) and is similar to the opercular cavity of the bony fishes, both being
water chambers outside the pharynx.The
atrium opens to the exterior through a largeatriopore(Fig
29-6).

Posterior
to the pharynx the gut narrows to form a shortesophaguswhich
connects the pharynx with thestomach(midgut).Anteriorly
the stomach bears a ventral anteriorly-directed diverticulum, the secretory and
absorptivehepatic cecum,
which pro­jects into the atrium on the right side of the pharynx (Fig 29-6).Students
with experience in vertebrate embryology will remember that the vertebrate liver
develops from a midgut diverticulum on the right side of the embryo.By
the structural, positional, and intermediate criteria the hepatic cecum of
amphioxus seems to be homologous to the liver of vertebrates.

The
stomach narrows posteriorly to become the darkeriliocolon.This
region of the gut tube bears a ring of ciliated epithelium that rotates the food
mass and mixes it with enzymes.Posterior
to the ring the gut continues as theintestine,
ultimately ending at theanus,
located at the base of the tail.The
asymmetrically located anus lies just to the left of the ventral fin.Thepostanal
tailextends
posteriorly from the anus.

3.
Amphioxus: pharyngeal cross section (Fig 3, 29-3C).With
low power of the compound microscope examine a slide of amphioxus cross sections
and find the section through the pharyngeal region.Slides
labeled "representative cross sections" will bear three or four sections taken
through different regions of the body.Make
sure you have the correct section (the one through the pharynx) by comparing it
with Fig. 3.Use
40X, 100X, and occasionally 400X, for this exercise.

Note
first the outerintegumentcompletely
surrounding the animal.Although
you cannot discern its composition, it consists of an outer acellular cuticle
which is secreted by the underlying epidermis.Below
the epidermis is the soft, connective tissue dermis.The
integument bulges outward along the dorsal midline where it covers thedorsal
fin.

Find
the numerousmyomeresalong
each side of the body and note that they are not bilaterally symmetrical (Fig
3).Each myomere is
surrounded by amyoseptumcomposed
of connective tissue.Dorsally
between the right and left muscle masses is a relatively smallnerve
cordand a much largernotochord.Both
are surrounded bysheathsof
connective tissue.

The
interior of the ventral half of the section is occupied by thepharynx.Itslumenis
surrounded by thegill bars,
appearing here in oblique section.In
living material the gill bars are nearly vertical but in most fixed specimens
the pharynx is distorted so that the bars are aligned obliquely or nearly
horizontally.As
a consequence you see them in oblique or cross section rather than sliced along
their long axis as you might have expected.Look
closely at the circle of gill bars noting first thegill
slitsthat separate them.It
is through these slits that water passes during feeding.Examine
some of the gill bars with 400X (careful!) and note the abundance ofciliaon
their inner margins.Lateral
cilia(Fig 3, 29-3C) are
located on the surfaces between the gill bars and generate the feeding current
which passes from the pharynx lumen through the gill slits to the atrium.In
contrastfrontal ciliaare
on the inner surface of the gill bars face the lumen and are responsible for
transporting food and mucus dorsally to the esophagus. Each gill bar is
associated with a small coelomic space and an aortic arch but these are
difficult to see.

Ventrally
along the pharynx there is a longitudinal, median, ciliated groove, theendostyle,
which secretes a copious mucus.The
mucus contains iodine and the endostyle is homologous to the vertebrate thyroid
gland (positional and intermediates criteria).Look
at the endostyle with high power to find its cilia.Ventral
to the endostyle there is a small endostylar coelom surrounding an even smaller
ventral aorta.The
endostylar coelom is homologous to the pericardial coelom of vertebrates.

Dorsally
in the pharynx there is another median furrow, theepibranchial
groove.The
paireddorsal aortaeare
located dorsolateral to it.Most
of the space surrounding the pharynx is theatrium,
which is enclosed by an ectodermal epithelial lining.

During
feeding the lateral cilia of the gill bars generate a water current that enters
the pharynx through the mouth.The
water passes laterally through the gill slits, out of the pharynx, and into the
surrounding atrium.Mucus
secreted by the endostyle is carried upward over the inner surface of the gill
bars by ciliary currents generated by frontal cilia.The
mucus entangles food particles attempting to pass between the gill bars and
transports them to the epibranchial groove.The
cilia of the groove then move the mucus and trapped food posteriorly into the
esophagus.Food is
moved through the gut by its ciliated walls. The epithelium of the hepatic cecum
secretes digestive enzymes into the gut lumen.Digestion
occurs extracellularly in the stomach and iliocolon and its products are
absorbed by the epithelium of the cecum.

Although
inconspicuous, the cephalochordatecoelomoccupies
its expected position surrounding the gut (Fig 29-3C, 29-9).Fairly
large perivisceral coelomic spaces may be seen lying dorsolateral to the
pharynx.Extensions
of this part of the coelom extend to the gill bars and are continuous with the
coelom in those structures.

Paired
segmental nephridia, which you may not see, are located in the perivisceral
coelomic spaces beside the pharynx (Fig 29-9).These
unusual nephridia have podocytes like metanephridia but also have a flagellated
collar cell and weir basket like protonephridia. The podocytes are associated
with the dorsal aorta and form an ultrafiltrate of the blood into the
perivisceral coelom.To
leave the coelom and enter the atrium for elimination, the urine must pass
through the weir basket of a flagellated collar cell.

The
ventrolateral regions of mature specimens are occupied by largegonads,
either ovary or testis (Fig 29-3A,B).Determine
the sex of your specimen by examining the contents of its gonads.Eggs
will be large and easy to see, sperm very small and unrecognizable. What is the
sex of your specimen?___________.You
are responsible for recognizing gonads of both sexes. The gonads are enclosed in
narrow coelomic spaces which may be difficult to see.

Note
thetransverse muscleventral
to the gonads.Themetapleural
foldsare folds of the body
wall and they contain a coelomic space, the pterygocoel (=metapleural coelom,
Fig 3, 29-3C).Thehepatic
cecum, enclosed in a small coelomic space, may be seen in cross section
beside the pharynx.Posterior
to the pharyngeal region the coelom is larger and surrounds the intestine.

The
pattern of blood flow in amphioxus is similar to that of early verte­brates
although there is no distinct heart (Fig 29-8).The
ventral aorta in the pharyngeal region gives rise to numerous pairs of aortic
arches that pass dorsally through the gill bars.Upon
leaving the gills the arches unite to form a pair of longitudinal dorsal aortae
dorsal to the pharynx.The
dorsal aorta carries blood posteriorly to muscles and gut.Blood
is returned to a ventral sinus venosus under the pharynx by a system of anterior
and posterior cardinal veins (which are not evident on these slides).The
sinus venosus is formed by the junction of the cardinal veins.The
ventral aorta extends anteriorly from the sinus venosus.

There
is also a visceral venous drainage system associated with the gut and hepatic
cecum that is probably homologous to the hepatic portal system of vertebrates.Blood
leaves capillaries in the gut wall via the hepatic portal vein that carries it
to capillary beds in the cecum.From
there the hepatic vein transports it to the sinus venosus. An hepatic portal
system is a universally present feature of vertebrates.

The ventral aorta is
contractile in amphioxus and functions as the heart but there are also tiny
pulsatile vesicles in the aortic arches.The
blood is acellular and unpigmented and probably plays no significant role in gas
transport.Hemoglobin
is, however, present in muscles and notochord (which is muscular).The
vessels lack an endothelial lining (like other invertebrates but unlike
vertebrates).